304
A. A. HORNER AND R. A. MORTON
The present work with castrates has shown that
the effect of cholesterol in males is not controlled by
their secondary sex hormones. However, it is of
primary importance to elucidate the precise
mechanism of the cholesterol-vitamin A effect in
male rats before attempting to discover why it is
not manifested in females.
SUMMARY
1. The effects of a dietary supplement of cholesterol on liver vitamin A stores have been determined for normal and castrated young male rats.
2. When vitamin A and cholesterol were given
in the diet together, liver vitamin A stores were
significantly lowered to the same degree in normal
and castrated animals. No significant changes in
kidney or plasma vitamin A levels were observed.
3. If the animals were given large doses of
vitamin A before cholesterol was given in a vitamin
A-free diet, liver vitamin A stores were not
affected in either normal or castrated rats.
I960
4. Castration significantly decreased the rate
of utilization of liver vitamin A in young male
rats.
5. In castrates receiving a cholesterol-free diet
liver unsaponifiable material was markedly reduced.
We thank Professor R. G. Harrison for castrating the
rats. The present study formed part of work which was
assisted by a grant from the Nuffield Foundation.
REFERENCES
Booth, V. H. (1952). J. Nutr. 48, 13.
Bult, A. R. & Sorgdrager, C. J. (1938). Acta brev. neerl.
Phy8iol. 8, 114.
Cama, H. R., Collins, F. D. & Morton, R. A. (1951).
Biochem. J. 50, 48.
Glover, J., Goodwin, T. W. & Morton, R. A. (1947).
Biochem. J. 41, xlv.
Green, B., Horner, A. A., Lowe, J. S. & Morton, R. A.
(1957). Biochem. J. 67, 235.
Moore, T., Sharman, I. M. & Ward, R. J. (1951). Biochem. J.
49, xiii.
The Chemical Composition of Snail Gelatin
BY A. P. WILLIAMS
The British Gelatine and Glue Research Association, 2a Dalmeny Avenue, London, N. 7
(Received 7 July 1959)
Early wor&k on the.-distribution of collagen in the
EXPERIMENTAL
animal kingdom, mainly based on X-ray studies,
was considered' by Bear (1952) and Rudall (1955).
Material8
Watson.(1958bj has reviewed the chemical analyses
Snail gelatin. The 200 mg. (air-dried weight; moisture
available for invertebrate collagens, which at that 12-4 %) sample of gelatin used was prepared from the body
time were -restricted to the cuticle of earthworm walls of 30 garden snails by Melnick (1958). The snails
(Lumbriew8 sp.) (Watson, 1958a), the cuticle of were killed by immersion in aqueous 10% (v/v) ethanol for
Allobophora longa (Singleton, 1957), the byssus 2 hr. The shells were removed and each body was opened by
threads of Mytilus eduli8 (Jackson et al. 1953), and cutting along the dorsal ridge from the mouth to the collar.
the spongin fibrils from the mesogloea of sponges The viscera were removed and the body walls soaked in 5 %
(w/w) hydrochloric acid to coagulate the mucus. The
(Gross, Sokol & Rougvie, 1956).
was then soaked in 0*5 % sodium chloride solution
Since Watson's review, analyses have appeared material
for a week followed by a short treatment in saturated
of the cuticle of Ascari8 lumbric,oidea, and of the lime water. The material was extracted by warming in
cuverian tubules of sea cucumber (Holuthuria water for 2 hr. at 700, 800 and 900. Each extract was
forskali), both by Watson & Silvester (1959), and a filtered and concentrated in vacuo over phosphorus pentpreliminary study was made of the body wall of the oxide. The gelatin used for analysis was that extracted
garden snail (Helix a8per8a) by Melnick (1958). at 900.
More recently, Piez & Gross (1959) have reported
Analytical method8
the analyses of Thyone body wall (Echinodermata),
(100 mg.) was hydrolysed with
The
Hydroly8i&.
of Metridium skin and Phy8alia float (Coelenterata), 20 ml. of 20% (w/w)gelatin
hydrochloric acid at 1000 in a sealed
and of spongin A and spongin B from Spongia tube for 24 hr.
graminae (Porifera). This present paper is concerned
Amino acid analysi8. The analysis was carried out by the
with the chemical composition of gelatin derived method of Moore & Stein (1951) with the modification
from the intact collagen of the body wall of the suggested by Eastoe (1955); the corrections applied were
those used by Eastoe. Hydroxyproline was estimated as by
garden snail.
Vol. 74
3C5
SNAIL GELATIN
Table 1. Amino acid composition and related analytical data for snail gelatin
Amino acid
Alanine
Glycine
Valine
Leucine
Isoleucine
Proline
Phenylalanine
Tyrosine
Serine*
Threonine*
Cystine
Methioninet
Arginine
Histidine
Lysine
Aspartic acid
Glutamic acid:
Hydroxyproline
Hydroxylysine
Amide N§
Total
Glucosamine
Amino acid N
(g./100 g. of
dry, ash-free
as % of
sample)
(moles/105 g.)
total N
5-69
63-8
5-82
21-24
24-95
283-1
2-22
1-75
18-97
2-07
3-02
20-74
1-40
10-64
0-97
10-57
91-8
8-34
1-45
8-75
0-78
1-41
7-78
0-71
54-2
4-92
5-70
24-4
2-2
2-90
0-00
0-00
0-00
1-07
0-16
0-13
16-43
45-6
7-88
2-33
0-65
0-36
7-30
1-33
1-07
58-9
5-37
7-84
87-4
7-89
12-85
87-7
11-50
7-95
7-2
1-29
1-14
49-2
4-46
0-69
930-9
99-09
0-98
0-06
0-17
0-26
2-66
0-48
12-10
12-75
15-47 (g./100 g. of dry, ash-free material)
93-6
94-7
98-3
12-4
0-76 (on air-dried material)
No. of residues
of amino acid
per 1000 total
residues of
amino acids
72-3
321-0
21-5
23-5
12-1
104-1
9-9
8-8
61-4
27-7
0-00
1-2
50-9
2-6
8-3
66-8
99-1
99.5
8-2
Galactosamine
Hexose
Total
Total N
Mean residue wt.II
Recovery by wt. (%)
Recovery of N (%)
Moisture (%)
Ash (%)
Corrected for decomposition during hydrolysis (Eastoe, 1955).
t Sum of methionine and methionine sulphoxide peaks.
$ Corrected for decomposition on the column (Moore & Stein, 1951).
§ Corrected for ammonia formed by decomposition of threonine and serine (Eastoe,
11 Chibnall (1942).
I
Neuman & Logan (1950). Total nitrogen was estimated by
the Kjeldahl method.
Moisture. The loss of weight on drying the gelatin at 1050
for 24 hr. was used to calculate the moisture content.
Ash. The gelatin was ignited with concentrated sulphuric
acid in a platinum dish, then heated at 550° to constant
weight and the residue weighed as sulphated ash.
Estimation of heXose. Hexose was estimated by the
anthrone reaction (Dreywood, 1946), with the modification
of Bangle & Alford (1954). The hexose is expressed as
galactose, this sugar being used for the preparation of the
standard curve.
Estimation of hexosamine. Glucosamine and galactosamine were estimated directly from the peaks on the amino
acid chromatograms as suggested by Eastoe (1954). Watson
(1958a) reports that this method gives rather low and
variable values but there was insufficient material available
for the indepcndent determination by another method.
Estimation of pentose. Pentose was estimated by the
orcinol reaction (Mejbaum, 1939), with the modification of
Albaum & Umbreit (1947). The pentose was expressed as
arabinose, this sugar being used for the preparationofthe
standard curve.
RESULTS
A summary of the amino acid composition and
related analytical data is given in Table 1. The
number of residues of a given amino acid per 1000
total residues, calculated as by Eastoe & Leach
(1958), is also given to enable comparisons to be
made with other gelatins and collagens.
When the gelatin was hydrolysed for the determination of amino acids a considerable amount of
insoluble humin was formed and the hydrolysate
was dark in colour. This is contrary to the observations made by Melnick (1958), who obtained lightcoloured hydrolysates and assumed the gelatin
samples to be largely free of polysaccharide.
Bioch. 1960, 74
306
A. P. WILLIAMS
The hydrolysate was filtered to remove the
humin and the analysis was carried out on the
filtrate. The presence of humin suggested large
amounts of polysaccharide components (Watson &
Silvester, 1959). The presence of polysaccharide
was confirmed by the detection of hexosamines,
common constituents of mucopolysaccharides, on
the amino acid chromatograms, both galactosamine
and glucosamine being detected on the short
column of the amino acid separation (Eastoe,
1954), and also by the detection of a peak of red
material at about fraction 42 on the long column of
the amino acid separation, indicating the emergence of carbohydrate-decomposition products
(Dustin, Czajkowska, Moore & Bigwood, 1953).
DISCUSSION
A common chemical feature of gelatins derived
from vertebrate and invertebrate collagens is the
high number of glycyl residues. This number, 321
in every 1000 amino acid residues, was also found
in snail gelatin. A number of chemical features
characteristic of vertebrate gelatins were found.
The prolyl and hydroxyprolyl contents are of the
same order; there is one hydroxylic side chain in
every five residues compared with one in six in
vertebrates.
.The paost striking feature, however, is the
presence of hydroxylysyl residues. The hydroxylysine contet isisifact higher than that of most
vertebrate cltlag6is nd gelatins. Eastoe & Leach
(19&3) s*mypJred fth-nino acid analyses available
for dte*c6llan and gelatin and found that
thosesampfes oniX-ch hydroxylysine estimations
had been oarrie&b,oUt all contained hydroxylysyl
residues in aifiounts varying from 3-5 to 12-2
residues per 1000 total residues. Of the invertebrate collagens and gelatins reviewed by Watson
(1958b) only the collagen derived from the cuverian
tubules of the sea cucumber had been found to
contain hydroxylysyl residues (4.7 per 1000 total
residues). Piez & Gross (1959), however, detected
hydroxylysine in all the invertebrate gelatins that
they studied, Metridium, Phyaalia and spongin B
having 25, 30 and 24 hydroxylysyl residues per
1000 residues respectively, the highest yet reported
for any collagen or gelatin. The amount found by
them in Thyone gelatin and spongin A, 11 and 12
residues per 1000 residues respectively, is of the
same order as that found in snail gelatin.
Although snail gelatin is similar to vertebrate
gelatins in amino acid composition it resembles the
invertebrate gelatins in the low total nitrogen
content. This may be explained by the presence of
considerable amounts of polysaccharide. The
polysaccharide distribution is comparable with that
found in some other invertebrate collagens and
I960
gelatins (Watson, 1958a; Watson & Silvester,
1959), hexoses being present in considerable
amounts together with a smaller amount of hexosamines. A small amount (0-66 g./100 g. of dry, ashfree material) of pentose was also estimated, but by
the orcinol method, which is not entirely specific.
There was insufficient material left to confirm the
presence of pentose or for the determination of
uronic acids.
Generally, vertebrate collagens and gelatins have
low polysaccharide contents. The cuticle of Ascari8
lumbricoide8 (Watson & Silvester, 1959) is similar in
this respect, but collagens from earthworm cuticle
(Lumbricu8 sp.; Watson, 1958a) and the cuverian
tubules of Holuthuria for8kali (Watson & Silvester,
1959) contain large amounts of polysaccharide.
There is insufficient evidence available to show that
the polysaccharide found in these materials and in
snail gelatin forms an integral part of the collagen
from which they were derived.
SUMMARY
1. A gelatin fraction isolated from the intact
collagen of the body wall of Helix a8per8a was
submitted to chemical analysis.
2. The analysis showed that the gelatin contains a high proportion of glycyl and hydroxyprolyl
residues. The proportion of hydroxyproline is
similar to that of proline. There are also a number
of hydroxylysyl residues present. The amino acid
composition of snail gelatin resembles vertebrate
gelatins more closely than the invertebrate gelatins
previously studied.
3. The remainder of the gelatin is polysaccharide and yields galactose and smaller amounts of
pentose and hexosanines on hydrolysis.
The author is indebted to Mr S. C. Melnick for the sample
of snail gelatin. The author is very grateful to Mr A. A.
Leach of The British Gelatine and Glue Research Association for advice on the Moore & Stein (1951) technique and
for much valuable discussion.
This paper is published by permission of the Director and
Council of The British Gelatine and Glue Research Association.
REFERENCES
Albaum, H. E. & Umbreit, W. W. (1947). J. biol. Chem.
167, 369.
Bangle, R. & Alford, W. C. (1954). J. Histochem. Cytochem.
2, 62.
Bear, R. S. (1952). Advanc. Protein Chem. 7, 69.
Chibnall, A. C. (1942). Proc. Roy. Soc. B, 181, 136.
Dreywood, R. (1946). Indlutr. Engng Chem. (Anal.), 18,
499.
Dustin, J. P., Czajkowska, C., Moore, S. & Bigwood, E. J.
(1953). Analyt. chim. acta, 9, 256.
Eastoe, J. E. (1954). Nature, Lond., 173, 540.
Eastoe, J. E. (1955). Biqphem. J. 61, 589.
Vol. 74
307
SNAIL GELATIN
Eastoe, J. E. & Leach, A. A. (1958). Recent Advances in
Gelatin and Glue Research, p. 173. Ed. by Stainsby, G.
London: Pergamon Press Ltd.
Gross, J., Sokol, Z. & Rougvie, M. (1956). J. Histochem.
Cytochem. 4, 227.
Jackson, S. F., Kelly, S. C., North, A. C. T., Randall, J. T.,
Seeds, W. E., Watson, M. & Wilkinson, J. R. (1953).
Nature and Structure of Collagen, p. 106. Ed. by Randall,
J. T. London: Butterworths.
Mejbaum, W. (1939). Hoppe-Seyl. Z. 258, 117.
Melnick, S. C. (1958). Nature, Lond., 181, 148.
Moore, S. & Stein, W. H. (1951). J. biol. Chem. 192, 663.
Neuman, R. E. & Logan, M. A. (1950). J. biol. Chem. 184,
299.
Piez, K. A. & Gross, J. (1959). Biochim. biophys. Acta, 84,
24.
Rudall, K. M. (1955). Symp. Soc. exp. Biol. 9, 49.
Singleton, L. (1957). Biochim. biophys. Acta, 24, 67.
Watson, M. R. (1958a). Biochem. J. 68, 416.
Watson, M. R. (1958b). Recent Advances in Gelatin and
Glue Research, p. 179. Ed. by Stainsby, G. London:
Pergamon Press Ltd.
Watson, M. R. & Silvester, N. R. (1959). Biochem. J. 71,
578.
A Native Cobalamin-Polypeptide Complex from Liver:
Isolation and Characterization
BY A. HEDBOM
In8titute of Biochemi8try, University of Upp8ala, Upp8ala, Sweden
(Received 8 June 1959)
In mammalian liver vitamin B12 and related substances occur mainly as weakly bound protein or
polypeptide complexes. These bound forms should
probably be regarded as the biologically functional
units rather than the free cyanocobalamin (Smith,
1958). Very little information, however, is as yet
available about the nature of these complexes.
In a preliminary note from this Laboratory, the
isolation of a cobalamin polypeptide from a liver concentrate (Organon WBC) was described (Hedbom,
1955). Because further supplies of this particular
raw material were not available, we have now
developed a method for obtaining a similar concentrate from fresh ox liver. From this it has been
possible to isolate a cobalamin polypeptide, with
essentially the properties previously described, in
quantities which enable us to perform a more
detailed examination.
This paper deals mainly with the isolation procedure and characterization of the cobalamin
polypeptide.
EXPERIMENTAL AND RESULTS
Isolation of the cobalamin-polypeptide
complex
No method of assay which is specific for the vitamin B12
conjugates in question is yet known. Therefore, the isolation could be guided only by determination of the total
vitamin B12 content.
In the starting material and the first fractions from the
isolation procedure, the vitamin B12 activity was detected
and estimated by microbiological assay. Various extraction
methods were tested in order to find the optimum conditions for the liberation of cyanocobalamin from the
samples without destruction, and different micro-organisms
requiring vitamin B12 were used and compared for the
estimation. A modification of Burkholder's (1951) Escherichia coli tube method was found to be satisfactory for the
purpose.
In more concentrated fractions the vitamin B12 content
was estimated by absorption spectrophotometry, or by
cobalt determination, as a check on the microbiological
determination.
Microbiological assay
Preparation of samples. The vitamin B12 was released
from the protein moiety by papain digestion. A sample
(1 g.) was suspended in 10 ml. of 0 1M-sodium acetate
buffer, pH 5.0, containing 50 mg. of papain (E. Merck,
Darmstadt) and a trace of cyanide. The mixture was incubated for 3 hr. at 400 and subsequently diluted with water
to a total volume of 50 ml. After filtration and appropriate
dilution, samples of the solution were assayed for vitamin
B.2 activity as described below.
Test organism. This was Escherichia coli mutant 113-3
(Davis & Mingioli, 1950). Stock cultures were stored and
the inoculae prepared as described by Burkholder (1951).
Basal medium. The medium (in fivefold concentration)
contained, in 200 ml. of water: K2HPO4 7-0 g., KH2PO4
3-0 g., trisodium citrate 0-5 g., MgSO4,7H20 0-1 g.,
(NH4)2SO4 1-0 g., glucose 10-0 g., thiomalic acid 0-1 g.,
L-asparagine 4-0 g., L-arginine 0-1 g., L-glutamic acid
0-1 g., glycine 0-1 g., L-histidine 0-1 g., L-proline 0-1 g.,
DL-tryptophan 0-1 g.
The pH was measured with a Beckman glass-electrode
pH meter, and adjusted, if necessary, to 7-0, by addition of
KOH. In each tube 1 ml. of medium was used.
Vitamin standard. This was a water solution containing
0-200,umg. of cyanocobalamin/mi., prepared fresh before
each assay from a stock solution containing 2 tLg. of cyanocobalamin (on a colorimetric basis) and 2 mg. of KCN/ml.
The standard solution was added in duplicates to assay
tubes in the volumes 0, 0-2, 0-5, 1-0, 1-5, 2-0 and 4-0 ml. per
20-2